1. Field of the Invention
The present invention relates to a device and a method for introducing a particle into a cell as well as a device and a method for collecting a particle out of a cell.
2. Related Background Art
P. J. Robinson et al. (Biotechnol. Bioeng., 15, 603-606 (1973)) have paid attention to the fact that the fine magnetic particles can be collected in a simple operation and reported in their research paper that fine magnetic particles may be used as an enzyme immobilization carrier for a bioreactor. In the research report, they immobilize α-chymotrypsin or β-galactosidase onto iron oxide fine particles or cellulose/iron oxide composite particles and apply such magnetic carrier particles to a complete mixed-type bioreactor. They demonstrate that such magnetic carrier particles can be easily aggregated or separated by magnetic means.
Japanese Patent No. 2642025 discloses that fine magnetic particles (for example, having an average particle size of 0.3 μm with a density of 5.2 g/cm3) having a biosubstance immobilized thereon are shot into cells at a high speed (initial speed: 50 to 400 m/second) by a particle gun method, thereby introducing the biosubstance, and that the cells having the fine magnetic particles introduced therein can be selectively concentrated and separated by magnetic means. Furthermore, Japanese Patent Application Laid-Open No. 2003-325176 discloses that the efficiency of introducing particles can be improved by a particle gun method using fine composite particles having a dense magnetic substance as their cores. However, since these methods fundamentally employ a particle gun method, many cells die by mechanical shock of fine particles applied upon the cells at a high speed, with the result that the total number of cells to which fine magnetic particles are successfully introduced therein decreases. Besides this, a cell introduction step and separation step cannot be supported out continuously and a device for supporting out these steps may become large scale. These problems still remain unsolved.
On the other hand, an electroporation method is known for introducing a gene into a cell. A foreign gene is introduced into a cell by electrically stimulating the cell membrane, thereby increasing its permeability. Since the gene introduction efficiency of this method is relatively good compared to a cell fusion method as well as a particle gun method, the electroporation method has been widely used. Japanese Patent Application Laid-Open No. 11-056360 discloses a technique for applying an electric field lower in strength than a conventional electroporation method, a larger number of times, while rendering the length of an electric pulse longer. More specifically, it is disclosed that a foreign gene is introduced directly into a plant cell (without applying any pretreatment (protoplastization) to the plant cell) by applying an electric field having a strength of 50 to 500 V/cm, 5 to 25 times while controlling the length of an electric pulse at 30 to 200 msec.
As a method of collecting a target biomolecule out of a cell, methods of killing and disintegrating cells (by use of physical disintegration and autolysis of cells) are generally known. However, these methods have problems that although it is necessary to separate and purified target cells by any means in advance, the purity of a target substance is low due to the presence of contaminants.
Japanese Patent Application Laid-Open No. 2004-49105 discloses a method of selectively and exclusively separating cellular constitutional components such as DNA from target cells by use of fine magnetic particles by selectively killing and disintegrating the target cells.
In any one of these methods, cells are disintegrated to collect target biomolecules.